Vascular transit time is an important indicator of microcirculatory health. We present a secondorder-plus-dead-time (SOPDT) model for robust estimation of kinetic parameters characterizing microvascular bolus passage using two-photon fluorescence microscopy (2PFM) in anesthetized rats receiving somatosensory stimulation. This methodology enables quantification of transit time, time-to-peak, overshoot, and rate of bolus passage through the microvascular network. The overall transit time during stimulation, of 2.2±0.1 seconds, was shorter (PB0.0008) than that at rest (2.7 ± 0.2 seconds). When compared with conventional c-variate modeling, the SOPDT modeling yielded better quality of fit both at rest (P < 0.0001) and on activation (P < 0.001).